Neutrophils serve as the body's primary cellular defense against bacterial infection. One of the mechanisms by which neutrophils destroy invading microorganisms is through the generation of various toxic oxygen metabolites via the so-called "respiratory burst" (Babior, NEJM 298: 659-668 (1978)). "Respiratory burst" is the name given the phenomenon that occurs when neutrophils undergo a large burst in respiration in which oxygen is converted to superoxide anion (O.sub.2 -), the initial product of the respiratory burst. Superoxide anion is generated by an NADPH oxidase found in neutrophils and other phagocytes (Babior, supra (1978; Clark, J. Infect. Dis. 161 : 1140-7 (1990)). This "enzyme" is actually a miniature electron transport chain consisting of multiple plasma membrane- and cytosollocalized protein components. The oxidase is apparently dormant in resting neutrophils, but acquires catalytic activity when the cells are stimulated. (See Curnutte, et al., J. Biol. Chem. 262: 6450-2 (1987).) This dramatic increase in oxidative metabolism triggered by phagocytosis or exposure to certain inflammatory mediators is also characteristic of mononuclear phagocytes and eosinophils, but it is best understood in neutrophils. (See Clark, J. Infect. Dis. 161: 1140-7 (1990).)
The importance of the NADPH oxidase for the neutrophil's antibacterial capacity is evidenced by patients with the inherited disorder chronic granulomatous disease. The neutrophils of patients with this disorder are unable to generate superoxide anion and are subject to persistent, severe bacterial infections, which often result in life-threatening episodes or even death (Clark, supra (1990); Curnutte, in Phagocytic Defects II: Abnormalities of the Respiratory Burst, Hematology/Oncology Clinics of North America, 241-252 (1988)). It has been shown that several forms of this disease result from genetic defects in one of the various protein components of the NADPH oxidase system (Curnutte, suora (1988)).
The mechanism by which the NADPH oxidase is activated by inflammatory stimuli is not well understood but appears to involve the assembly of the various components of the NADPH oxidase at the plasma membrane level to form an "active" complex (Clark, suora (1990)). The processes involved in the translocation of cytosolic oxidase components to the membrane also remain to be defined. There is evidence that a GTP-binding protein is involved in regulating the activation process (Quilliam and Bokoch, in Cellular and Molecular Mechanisms of Inflammation, Vol. 2 (1991); Cochrane and Gimbrone, eds., Academic Press, San Diego, CA). Indeed, a GTP-binding protein known as RaplA (see Quilliam, et al., Mol. Cell. Biol. 10: 2901-8 (1990)) has been shown to bind to the cytochrome b component of the NADPH oxidase (Quinn, et al., Nature 342: 198-200 (1989); Bokoch, et al., Science 254: 1794-6 (1991)). Rac2 has now been identified as a stimulatory regulator of the oxidase in human neutrophils (See Knaus, et al., Science 254: 1512-1515 (1991).
The low molecular weight GTP-binding proteins (LMWG) represent a rapidly growing superfamily of GTPases that regulate a wide variety of cellular processes (Hall, Science 249: 635-40 (1990)). These proteins consist of a GTP-binding monomer with a molecular weight of 19,000-28,000 and have properties that distinguish them from the various receptor-coupled G protein .alpha. subunits, including their lack of associated .beta./gamma subunits. Although the LMWG can vary greatly in their overall amino acid sequences, they exhibit a number of features that are common to each. These include (1) common structural motifs; (2) regulation by extrinsic factors that modulate whether the protein is in a GTP- or GDP-state; and (3) posttranslational processing by isoprenylation, proteolytic truncation, and carboxymethylation. The latter is directed by a CAAX consensus motif found at the carboxyl terminus of all known isoprenylated proteins, where C is a cysteine residue, A is any aliphatic amino acid, and X is variable (Maltese, FASEB 4: 3319-3328 (1990)). proteins that affect the guanine-nucleotide binding and hydrolysis activity of various LMWG have also been identified, including guanosine triphosphate activating proteins (GAPs), proteins that stimulate guanine nucleotide exchange, and proteins that inhibit guanosine diphosphate (GDP) dissociation. (See Bokoch, et al., Science 254: 1794-6 (1991) and references cited therein.)
Posttranslational processing involves an initial isoprenylation at the cysteine residue via a thioether bond between the protein and a C15 (farnesyl) or C20 (geranylgeranyl) isoprenyl moiety. This is followed by proteolytic truncation of the protein, removing the three amino acids distal to the isoprenylated cysteine. The newly-exposed COOH-terminal cysteine is then carboxymethylated. For the Ras proteins, each of these processing steps has been shown to be an important determinant of Ras binding to the plasma membrane (Hancock, et al., EMBO J. 10: 641-646 (1991]; Hancock, et al., Cell 57: 1167-1177 (1989)) and isoprenylation is critical for proper expression of the transforming activity of oncogenic Ras (Casey, et al., PNAS USA 86: 8323-8327 (1989); Schafer, et al., Science 245: 379-384 (1989); Jackson, et al., PNAS USA 87: 3042-3046 (1990)).
Various studies have identified a multiplicity of cellular proteins that appear to be covalently modified by isoprenyl groups (Maltese, supra (1990); Glomset, et al., Trends Biochem. Sci. 15: 139-142 (1990)). The electrophoretic patterns of these proteins is remarkably similar from one cell to another, and the proteins generally fall into two size classes. A group of 44-69 kD isoprenylated proteins are largely localized to the nucleus and the associated nuclear matrix. Within this group are the nuclear lamins (Maltese, supra (1990); Schafer, supra (1989); Wolda, et al., J. Biol. Chem. 263: 5977-6000 (1988)). A second class of 20-24 kD isoprenylated proteins are more widely distributed within the cell and appear to represent the LMWG (Maltese, et al., J. Biol. Chem. 265: 2148-2155 (1990)). A common feature of all known isoprenylated proteins is the presence of a CAAX motif at the carboxyl terminus. This sequence appears to represent a signal for protein isoprenylation and is present in most of the LMWG that have been identified (Maltese, FASEB 4: 3319-3328 (1990); Glomset, et al., Trends Biochem. Sci. 15: 139-142 (1990)).
The importance of O.sub.2 --in bacterial killing is evidenced by the chronic infections and even death observed in patients with severe neutropenia, chronic granulomatous disease, and other disorders of neutrophil function. However, the inappropriate or excessive formation of O.sub.2 --and its byproducts can both initiate and exacerbate inflammation. Inflammatory diseases and/or secondary inflammation resulting from a primary disorder are serious health problems. Therefore, the development of means to intervene in these processes in a specific manner is of great therapeutic interest. In addition, identification of key proteins involved in NADPH oxidase activation in phagocytic cells and the development of means to inhibit or otherwise regulate these proteins is of equal significance.